JP5430813B2 - Proline-accumulating yeast, method for producing the same, and method for producing sake using the yeast - Google Patents

Description

  The present invention relates to a proline-accumulating transformed yeast having excellent resistance to stress such as ethanol, freezing, and drying, a method for producing the same, and a brewed food or fermented food using the yeast, particularly a method for producing sake. More specifically, the present invention relates to a proline-rich transformed yeast that effectively accumulates proline in cells and secretes a part thereof extracellularly by genetic recombination, and a method for producing the same. The present invention relates to a method for producing sake with improved taste and flavor using yeast.

Fermented and brewed foods such as miso, soy sauce, sake, and bread are all applied to the fermentation or brewing action of yeast, and are now indispensable for daily life.
Of these, fermented food means food made using a fermentation reaction in which fermenting microorganisms such as mold, yeast, and bacteria decompose organic compounds to produce alcohols, organic acids, carbon dioxide, etc. A food means a food made by fermentation and aging, that is, a food made by brewing.
These fermented foods and brewed foods range from seasonings such as miso, soy sauce and vinegar, alcoholic beverages such as sake, beer, shochu, whiskey, pickles, natto, bread, cheese, yogurt, and lactic acid bacteria beverages. Even if the same raw materials are used, different foods are produced if different fermentation microorganisms are used. In the case of miso and sake made from rice, the process is the same until the koji is made using koji mold, but then it becomes miso with the help of lactic acid bacteria, and it becomes kake using yeast. Furthermore, after alcohol fermentation, if brewed using acetic acid bacteria, it becomes vinegar.
In recent years, especially as life has become richer, a variety of luxury products have been developed, and the differentiation and individualization of taste and fragrance is becoming increasingly popular. This is no exception in the field of brewing, especially in the liquor field. There is a growing tendency to demand a variety of flavors and aromas that are also the life of refined products in sake. The taste of sake is formed by the interaction of various components, but is greatly influenced by the composition of organic acids and amino acids. In general, amino acids in sake are considered to be the cause of the “miscellaneous taste” of sake, and some people think that they should be reduced. However, as is apparent in soy sauce and miso soup, there are no problems with amino acids themselves. It is considered that amino acids can be a cause of “miscellaneous taste” and a component of “umami” depending on the balance with the taste components, particularly sugar and acid. Since amino acids in sake are mainly synthesized by yeast, in order to respond to the diversification of the taste of sake, yeast that can control the organic acid composition related to taste while maintaining the alcohol-producing ability of sake yeast It is important to breed yeasts characterized by amino acid composition and production.

Yeast is classified into approximately 60 genera and 500 species, and the classification system is as follows.
1. Spore yeast (33 genera, 183 species);
Picha; 56 species Hansenula genus; 30 species Zygosaccharomyces genus; 8 species Saccharomyces genus; 7 species Basidiomycetous yeast (10 genera, 36 species)
3. Incomplete yeast (17 genera, 281 species)
Candida; 196 species

  The “strains” that use or apply these yeasts are all different for each food, and therefore all fermented and brewed foods have a unique flavor. These yeasts are named according to their intended use, such as “sake yeast”, “alcoholic yeast”, “beer yeast”, “vinegar yeast”, “baker yeast”, etc. Yes.

  These yeasts are subjected to stresses such as low temperature, freezing, drying, oxidation, high osmotic pressure, high alcohol concentration, and malnutrition in brewing or fermentation production environments, and many yeast cells are subjected to such stress for a long time. The inner protein is denatured, and there is a problem that useful functions of yeast are limited. For example, sake yeast has a problem that its productivity is reduced by stress caused by ethanol.

  In order to solve such problems, the present inventors have already focused on the fact that proline has the property of protecting yeast from stresses such as freezing, drying and oxidation. A patent application was made for baker's yeast (see Patent Document 1). The invention according to this patent application relates to a freeze-resistant yeast that accumulates one or more amino acids selected from proline, arginine, lysine, and glutamic acid in the fungus body, and a method for producing frozen bread dough and bread using the yeast. It is. In the present invention, in order to obtain a yeast that accumulates specific amino acids in the microbial cells, the parent strain is subjected to a mutagenesis treatment or a specific amino acid precursor is added to the medium separately from the carbon source necessary for yeast growth. Thus, a method for accumulating specific amino acids in the microbial cells was used.

  In addition, the inventors have accumulated a proline in a cell out of mutants exhibiting resistance to azetidine-2-carboxylic acid (AZC), which is a toxic analog of proline, and exhibiting refrigeration stress resistance than the parent strain. We succeeded in isolating the strain (FH515 strain) and reported it (see Non-Patent Document 1). In normal cells, the proline synthesis system is strictly regulated, and there is also a degradation system, so proline is not accumulated excessively in the cells. Therefore, even in wild yeast strains, it cannot grow in a medium containing AZC, which is a toxic analog of proline, but in cells with increased proline content in the cell, the AZC incorporated into the cell is relatively diluted and is resistant to AZC. I found out that

  Furthermore, the present inventors have mutated the gene (PRO1) encoding γ-glutamate phosphorylase (γ-GK) in the mutant strains mentioned in the above-mentioned literature, and Asp154 is substituted with Asn. (Refer to Non-Patent Document 2) Reported that stabilization of γ-GK increases both γ-GK and γ-glutamylphosphate reductase (γ-GPR) enzyme activities, resulting in excessive synthesis of proline. (See Non-Patent Document 3).

  The present inventors also show that when the gene for proline oxidase (PUT1), an enzyme involved in the first step of the proline degradation system, is disrupted, the intracellular proline content also increases and resistance to freezing and drought stress is improved. (See Non-Patent Documents 4 and 5).

Recent progress in genetic engineering technology is remarkable, and as an application to the breeding of microorganisms, the production of transformants by gene transfer (recombination) has been actively performed. This is no exception in the brewing field such as liquor, and as in the case of mutant strains, research on transformation by genetic manipulation of brewing yeast has been conducted in order to improve work efficiency and improve liquor quality such as taste and flavor. It is actively done. However, at present, there are no examples in which the gene recombination technology has been put to practical use for alcoholic beverages and yeasts used for brewing beer. In addition, umami, in particular, yeast that has the characteristics that the intracellular accumulation amount of proline, which is a cause of sweetness, is secreted to the outside of the cell, and that ethanol resistance and ethanol production ability can be improved, In particular, sake yeast has not been put into practical use yet, and even its existence has not been known.
Japanese Patent Laid-Open No. 9-234058 H. Takagi, F. Iwamoto, and S. Nakamori, Appln.Microbiol., Biotecnol., 47, 405-411 (1977) Y. Morita, S. Nakamori, and H. Takagi, Appln. Environ. Microbiol., 69, 212-219 (2003) Y. Terao, S. Nakamori, and H. Takagi, Appln. Environ. Microbiol., 69, 6527-6532 (2003) H. Takagi, K. Sakai, K. Morida, and S. Nakamori, FEMS Microbiol. Lett., 184, 103-108 (2000) Y. Morita, S. Nakamori, and H. Takagi, J. Biosci. Bioeng., 94,390-394 (2002)

  Therefore, an object of the present invention is to improve the ethanol tolerance while improving the intracellular accumulation of proline, which contributes to sweetness, and secreting a part of it to the outside of the cell, and at least maintaining the ability to produce ethanol. It is providing the yeast which has such a characteristic, especially sake yeast.

  More specifically, the object of the present invention is to disrupt proline-degrading enzyme involved in the first step of the degradation system, ie, prolinease (PO), in the yeast proline metabolic pathway, thereby destroying proline. On the other hand, a gene encoding γ-glutamate kinase (γ-GK), which is a proline synthase involved in the first step of the synthesis system, is replaced with a proline-rich gene. Thus, it is to provide a transformed yeast that effectively accumulates proline in the cell and releases a part thereof outside the cell, and a method for producing the same. Furthermore, another object of the present invention is to efficiently produce fermented or brewed foods with improved umami, particularly sake having various flavors and aromas, using such transformed yeast.

  The present inventor conducted intensive studies to solve the above-mentioned problems, and as a result, the proline degradation by destroying the gene encoding the proline degrading enzyme involved in the first step of the degradation system in the yeast proline metabolic pathway. The synthesis system is enhanced by substituting the gene encoding the proline synthase involved in the first step of the proline synthesis system with a proline multi-producing gene. The present invention has been completed by finding a novel yeast whose content is further increased and a part thereof is released to the outside of the cell. In addition, these transformed yeasts have been found to have excellent resistance to various stresses such as freezing, drying, oxidation and ethanol, in particular ethanol stress, and brewing or fermenting foods such as sake with improved flavor and aroma. By finding it beneficial to manufacture, the present invention has been completed.

  That is, the present invention relates to a proline-accumulating transformed yeast having excellent resistance to stresses such as freezing, drying, and ethanol, a method for producing the same, and uses thereof, and is specifically as follows. .

1. A method for producing a proline-accumulating transformed yeast having the ability to secrete a portion of proline accumulated in cells to the outside during brewing, using sake yeast comprising the following steps.
a) a step of suppressing a proline degrading system consisting of destroying a gene of a proline oxidase that is a proline degrading enzyme in a proline metabolic pathway in yeast,
b) In the yeast proline metabolic pathway, the step of strengthening the proline synthesis system consisting of substituting Asn of Asp154 of the gene encoding γ-glutamate phosphorylase, a proline synthase, and c) proline in the cell A process of selecting and separating the accumulated strains.
2. A proline-accumulating transformed yeast XUDput1-MT (FERM P-20171) obtained by the method described in 1 above.
3. A method for producing sake, wherein the brewing is performed using the proline-accumulating yeast according to 2 above.

  The proline-accumulating yeast according to the present invention not only has a significant increase in the amount of proline inside and outside the cell, but also has a function of secreting a part of it to the outside of the cell, so that it can be cooled, frozen, dried, oxidized, and hyperosmotic. In addition to having resistance to stress such as malnutrition, it also has excellent resistance to high ethanol concentrations. From this, by using the yeast of the present invention, sake with excellent flavor and flavor can be produced efficiently.

  The yeast used in the present invention is not particularly limited as long as it has genes encoding both the prolylase (PO) as a proline-degrading enzyme and the γ-glutamate kinase (γ-GK) as a proline synthase. Rather, the desired yeast can be obtained by disrupting and replacing these genes. However, yeast belonging to the genus Saccharomyces cerevisiae is preferred. More specifically, Saccharomyces genus cerevisiae including sake yeast (Association No. 7 yeast, Association No. 9 yeast, Association No. 10 yeast, etc.), shochu yeast, wine yeast, beer yeast, baker's yeast and other practical yeasts. Yeast belonging to (cerevisiae), particularly preferably sake yeast belonging to the genus Saccharomyces cerevisiae.

  The proline synthesis pathway and its degradation pathway of yeast belonging to the genus Saccharomyces cerevisiae are as shown in FIG. In the figure, italic letters in parentheses indicate the names of genes encoding each enzyme. The proline synthesis system is the first step in which glutamate, which is a raw material, becomes γ-glutamyl phosphate (γ-GP) by the action of γ-glutamate kinase (γ-GK), and γ-GP is a γ-GP reductase It consists of a second step in which glutamic acid-γ-semialdehyde and pyrroline-5-carboxylic acid (P5C) are converted by the action of (GPR), and a third step in which P5C is converted to proline by the action of P5C-reductase. On the other hand, the proline degrading system comprises a first step in which proline is converted to P5C by prophosphorylating enzyme (PO) and two steps in which the P5C is converted to glutamic acid by the action of dehydrogenase.

  The “prophosphate oxidase gene” is a gene encoding an enzyme that converts proline to P5C, and it is possible to suppress the degradation of proline by destroying (knockout) it.

  The “proline degrading enzyme gene” means a gene encoding a prophosphorylating enzyme in the proline degrading system shown in FIG.

  The “wild-type proline synthase gene” means a gene encoding wild-type γ-glutamate kinase (γ-GK) that acts in the first step of the proline synthesis system shown in FIG.

  The “proline-rich gene” specifically means a gene obtained by substituting the Asp154 site of a gene encoding wild-type γ-glutamate phosphorylase (γ-GK) with Asn.

  “Proline-accumulating yeast” is a gene that encodes proline-degrading proline oxidase in yeast and loses its degradation function, and the synthetic γ-GK gene is a proline-producing gene. This is a replacement gene.

  “Destruction” refers to genetic manipulation that causes loss or inactivation of the gene function, and is specifically performed by a homologous recombination method.

Next, a method for producing a transformed yeast that accumulates proline according to the present invention will be specifically described.
The production process of the proline-accumulating yeast of the present invention comprises the steps of a) suppressing the degradation system, b) strengthening the synthesis system, and c) proline-accumulating type in the yeast proline metabolic pathway shown in FIG. It can be roughly divided into the steps of selecting and separating transformed yeast.

a) Step of inhibiting degradation system First, as the degradation system inhibition step a), PUT1 encoding the gene of proline oxidase (prophosphorylating enzyme), which is the first proline degrading enzyme, is destroyed (FIG. 2).
Various methods for gene disruption have been reported, but it is preferable to use the homologous recombination method in that only a specific gene can be disrupted. Among homologous recombination, one-step gene disruption method (one-step gene disruption method) can be obtained from the viewpoint that a strain that does not revert spontaneously can be obtained and, as a result, a highly safe strain can be obtained. ) Is preferred.

  The destruction of PUT1 is accomplished by deleting most of the ORF of PUT1 cloned on the plasmid, inserting a selectable marker gene (TRP1) into it, and then introducing a linear DNA fragment into yeast. It is carried out by a method in which both ends of the fragment are recombined twice between homologous portions of PUT1 on the chromosome and replaced with a DNA fragment sandwiching TRF1. According to this method, a complete deletion mutation of the target gene can be obtained, and there is an advantage that a back mutation due to recombination of the inserted fragment does not occur.

  The length of the homology region at both ends of TRP1 may be 40 bases or more, preferably 100 bases or more, more preferably 500 bases or more. In addition, the homology between both ends is preferably 90% or more, more preferably 95% or more.

  That is, a DNA fragment in which most of the PUT1 gene is replaced with a selection marker gene is introduced into a haploid of sake yeast, and then a transformant is obtained by homologous recombination with PUT1 on the chromosome. Means for confirming the destruction of PUT1 in the transformant thus obtained include proline assimilation ability, PCR method (polymer chain reaction method), enzyme activity measurement and proline content measurement method, etc. .

b) Strengthening step of the synthetic system Next, as the reinforcing step b) of the synthetic system, the wild strain PRO1 is replaced with the mutant PRO1 using the PUT1-disrupted strain obtained in the above step a) (FIG. 3). Using a selectable marker gene of URA3, constructing a plasmid incorporating a mutant PRO1 and then introducing it into a PUT1-disrupted strain. By homologous recombination between the mutant gene PRO1 on the plasmid and the wild-type gene PRO1 on the chromosome, A transformant in which the entire length of the plasmid containing URA3 is inserted into the wild type PRO1 locus is obtained.

c) Selection and separation step of proline-accumulating transformed yeast Finally, the transformed yeast obtained in the above step b) is cultured in a YPD complete medium and 5-fluoroorotic acid (5-FOA) medium to obtain a wild type The region containing PRO1 is removed, and a proline accumulating strain (AZC resistant) in which only mutant PRO1 remains in the chromosome is isolated (FIG. 3).
That is, a plasmid containing wild-type PRO1 and URA3 is removed, and a URA3-deficient strain is selected using a 5-FOA medium that cannot grow due to the formation of toxic compounds in the presence of URA3. By further accumulating proline from the obtained 5-FOA resistant colonies, a strain exhibiting resistance to AZC (mutant PRO1) and a strain exhibiting sensitivity (wild-type PRO1) are selected. The base sequence of PRO1 of both strains is confirmed by direct sequencing.

  In this way, the present inventors succeeded in producing a yeast that accumulates proline in cells, and named this proline accumulating strain (AZC resistant) as XUDput1-MT. Deposited as FERM P-20171 at the Deposit Center.

  Moreover, about the proline accumulation type transformed yeast of this invention, the growth experiment in ethanol presence was conducted, and characteristics, such as ethanol tolerance of the transformed yeast, were examined. Furthermore, using the proline-accumulating yeast of the present invention, a small preparation experiment of sake was conducted, and the brewing specification was also examined.

  In the present invention, plasmid preparation from E. coli was performed using Wizard Plus Minipreps DNA purification system (Promega) based on the alkaline SDS method. Other genetic manipulations such as transformation of E. coli, DNA restriction enzyme ligation, and ligation are described in “Biomanual Series I: Basic Technology of Genetic Engineering” (Yodosha) and “Bio-Experiment Illustrated” (Shyujunsha). In addition, handling of yeast and gene manipulation were performed according to “Biomanual Series 10 Genetic Experiment Method Using Yeast” (Yodosha) and “Biochemical Experiment 39 Yeast Molecular Gene Experiment Method” (Academic Publishing Center).

  Hereinafter, the present invention will be specifically described using yeast Saccharomyces cerevisiae, but the present invention is not limited thereto.

   The experimental materials and test methods used in this example are as follows.

1. Strains and plasmids (1) Yeast Saccharomyces cerevisiae;
“MB strain” is a hybrid of Σ1278b and S288C strain, and “MB329-17C (MATα ura3-52 trp1 put1-54 azc ^s )” was sold by Dr. Marjorie Brandriss (UMDNJ-New Jersey Medical School, USA). I received it.
“FH515 (MATα ura3-52 trp1 put1-54 pro1 ^D154N AZC ^r )” is a proline-accumulating AZC-resistant mutant isolated from MB329-17C by treatment with ethylmethanesulfonic acid (EMS), and a PRO1 mutation (Asp154Asn) have.
“XUW-14 (MATα ura3 trp1)” is a haploid of sake yeast and was sold by Mr. Yoshito Kubo of Fukui Food Processing Research Institute.

(2) Escherichia coli;
・ DH5α;
F- λ- φ80lacZ ΔM15 Δ (lacZYA argF) U169 deoR recA1 endA1 hsdR17 (r _k -m _k ⁺ ) supE44 thi-1 gyrA96
・ JM109;
recA1 end1 gyr96 thi1 hsdR17 supE44 relA1 Δ (lac-proAB) / F '[traD36 proAB ⁺ laclq kacZΔM15]

(3) plasmid;
“PBlue-PUT1” is a plasmid in which an approximately 2.6 kb fragment containing the PUT1 fragment is incorporated into the SalI-SacI site of pBluescriptIISK + (Toyobo Co., Ltd.) containing the origin of replication in E. coli and the ampicillin resistance gene of the selectable marker. .

  “PRS414” (Stratagene) is a yeast centromere type plasmid (YCp type), a plasmid comprising the yeast selection marker TRP1, and the origin of replication in E. coli and the selection marker ampicillin resistance gene. is there.

“PRS-D154NPRO1” is a plasmid obtained by incorporating a fragment of about 1.8 kb containing the mutant PRO1 (pro1 ^D154N ) into the HindIII-SacI site of ^pRS414 .

  “PRS406” (Stratagene) is a chromosomal integrative plasmid (YIp type), which contains the selection marker URA3 in yeast and the origin of replication in E. coli and the ampicillin resistance gene of the selection marker. . It does not replicate autonomously in yeast and is maintained only by chromosome integration by homologous recombination.

  “PUV2 (distributed by Dr. Junichi Incheon of Kyushu Institute of Technology)” is a multi-copy plasmid (YEp type) having a 2 μm DNA replication origin of yeast, which is the selection marker URA3 in yeast and the origin of replication in E. coli. Contains the selectable marker ampicillin resistance gene.

  “PUV-PRO2” is a plasmid in which an approximately 3.4 kb fragment containing wild-type PRO2 is incorporated into the BamHI site of pUV2.

2. Medium The medium used is as follows.

(1) YPD medium (complete medium for yeast);
Glucose 2%
Polypeptone 2%
Yeast extract 1%
Agar (2%) was added as needed.

(2) SD medium (minimum medium for yeast);
Glucose 2%
Yeast nitrogen base w / o amino acids (Difco Laboratories) 0.67%
As required, substances required for each strain (Uracil 16 mg / L, L-Leucine, L-Histidine-HCl, L-Tryptophan 40 mg / L each) or agar (2%) was added.

(3) SD (-N) medium (medium excluding ammonium sulfate from SD);
Glucose 2%
Yeast nitrogen base w / o (NH4) 2SO4, amino acids (Difco Laboratories) 0.67%
If necessary, the nitrogen source was 0.1% proline or sodium glutamate, and agar (2%) was also added.

(4) SC medium (complete synthetic medium for yeast);
Glucose 2%
Yeast nitrogen base w / o amino acids (Difco Laboratories) 0.67%
Drop-out mixture 0.2%
The above “Drop-out mixture” is a mixture obtained by removing specific substances from the substances shown in Table 1 as necessary and mixing the rest well.

酵母の培養は、30℃で行い、液体培地ではアルミキャップを用いた試験管又はシリコ栓を用いた坂口フラスコを用いて培養した。

The yeast was cultured at 30 ° C., and the liquid medium was cultured using a test tube using an aluminum cap or a Sakaguchi flask using a silico stopper.

(5) LB medium (complete medium for E. coli);
Tryptone 1%
Yeast extract 1%
NaCl 0.5%
Ampicillin (Amp) (50 μg / ml) and agar (2%) were added as needed.

(6) 5-fluoroorotic acid (5-FOA) medium (for 500 ml);
As shown in Table 2.

Agarと水をオートクレーブ後（121℃、15分）、冷却してから他の成分を添加する。

After autoclaving Agar and water (121 ° C, 15 minutes), cool and add other ingredients.

Here, “10 × YNB” is as follows.
Yeast nitrogen base w / o amino acids (Difco Laboratories) 1.5%
Ammonium sulfate 5%

Here, “10 × HC-7aa” is as shown in Table 3.

3. DNA oligomer Table 4 shows the sequences of the DNA oligomers used in this experiment. The synthesis of each oligomer was outsourced to Hokkaido System Science.

下線部は各制限酵素の認識部位を示す。

The underlined portion indicates the recognition site of each restriction enzyme.

4). Test method (1) Measurement of intracellular proline content
(1) -1. Measurement of dry weight Absorbance (OD600) after culture was measured, and the dry weight of the cells was calculated using the formula x = y / 978.45 (x = dry weight per 5 ml (g), y = OD600).

(1) -2. Quantification with amino acid analyzer Each strain was cultured in 5 ml of SD medium at 30 ° C for 48 hours, then collected in a centrifuge (3,500 rpm) for 10 minutes, further washed, and washed with 0.5 ml of sterile water. After suspending the body, it was treated with hot water at 100 ° C. for 10 minutes. After centrifugation (12,000 rpm, 5 minutes), the supernatant is diluted 5 to 10 times with 0.02NHCl (but 50 times for sake), filtered, and amino acid analyzer (Hitachi: L-8500A) High pressure amino acid analyzer). As the standard, 1 ml of amino acid mixed standard solution (containing 2 nmol / 20 μl of each amino acid) was similarly filtered and used.

(2) Ethanol stress susceptibility test After each strain was cultured in 10 ml of YPD medium at 30 ° C for 48 hours with shaking (preculture), SD (9 ml) + preculture (1 ml) [ethanol final 0% (v / v)] SD + 10% ethanol (9 ml) + preculture (1 ml) [ethanol final 9% (v / v)], SD + 20% ethanol (9 ml) + preculture (1 ml) [ethanol final 18% (v / v)] Was prepared and allowed to stand at 30 ° C. for main culture. After 0, 2, 5, and 8 days, each culture solution was diluted, spread on a YPD plate, and cultured at 30 ° C. for 2 days. The number of colonies produced was counted, and the survival rate was calculated with the number of viable bacteria on day 0 of culture as 100%. In addition, the intracellular proline content was measured using 5 ml of the preculture.

(3) Measurement of proline oxidase activity
After culturing in 50 ml of SD liquid medium at 30 ° C. for 48 hours, the cells were collected on a filter and immersed in liquid nitrogen for 10 seconds.
Next, place the membrane filter in an ice-cooled assist tube containing 10 ml of HEPES buffer (pH 7.5) + 3 mM MgCl ₂ and place on ice. When all the samples are ready, stir and suspend at the maximum speed of the vortex mixer. An enzyme solution was used.
Next, 0.5 ml of the crude enzyme solution was placed in the microtube, 0.4 ml of 10% proline was added, and the mixture was incubated at 30 ° C. for 15 minutes. Thereafter, 0.1 ml of o-aminobenzaldehyde solution (Sigma) and 0.5 ml of 10% TCA were added and vortexed, and allowed to stand for 30 minutes until color was developed. After centrifugation (12,000 revolutions / minute, 5 minutes), the absorbance (OD443) of the supernatant was measured and used as the proline oxidase activity value.
The enzyme activity is the amount of enzyme that produces ¹ nmol of Δ ¹ -pyrroline-5-carboxylate (P5C) -o-aminobenzaldehyde complex (ε = 2.71 × 10 ³ M ^-1 · cm ^-1 ) ^per 30 min at 30 ° C. 1 unit. Formula 1 shows the calculation formula.

1. Production of proline-accumulating strains Sake yeast that accumulates proline is produced by mutating XUDput1 obtained by destroying PUT1 of XUW-14 (MATα ura3 trp1), which is a haploid of sake yeast, with TRIP1 and using PRO1 (pro1 ^D154N ) A proline-accumulating strain XUDput1-MT was obtained by substitution. Hereinafter, this will be specifically described in order.

a) Suppression process of degradation system: disruption of PUT1 gene As shown in Fig. 2, plasmid pBlue-PUT1 was cleaved with restriction enzymes Bal I and I Aat I and a part of ORF of PUT1 was removed by electrophoresis. (3.9 kb) was recovered, and a TRP1-containing fragment (2.6 kb) obtained by cleaving plasmid pRS414 (purchased from Stratagene) with restriction enzymes Nae I and Sca I was ligated thereto and then introduced into E. coli strain JM109. did. A plasmid was prepared from a transformant exhibiting ampicillin resistance and named pBlue-Dput1-TRP1.

  Next, the plasmid pBlue-Dput1-TRP1 was cleaved with restriction enzymes BamH I and Kpn I to recover a TRP1-containing fragment (3.6 kb). Using this fragment, a haploid XUW-14 strain of sake yeast was used. Transformation was performed by homologous recombination with PUT1 to obtain a PUT1 disruption strain (XUDput1) of sake yeast.

In order to confirm the destruction of PUT1, a fragment (3.6 kb) containing TRP1 obtained by cleaving the above plasmid pBlue-Dput1-TRP1 with restriction enzymes BamH I and Kpn I was recovered, and this fragment was used to obtain the XUW14 strain. After transformation using homologous recombination with PUT1, the cells were spread on SC-Trp plates and cultured at 30 ° C. for 2 to 3 days. The obtained Trp ⁺ colonies were cultured in SD (-N) + 0.1% sodium glutamate medium and SD (-N) + 0.1% proline medium, and grown on sodium glutamate medium, but not on proline medium. The destruction of PUT1 was confirmed by colony PCR.
More specifically, the confirmation of the destruction of PUT1 was confirmed by the following four methods.

1) Proline utilization ability;
In general, yeast can grow using glutamic acid or proline as the only N source, but it is known that a PUT1-disrupted strain cannot assimilate proline. Therefore, in this experiment, each strain refreshed on SD agar medium was streaked to 0.1% sodium glutamate (MSG) or agar medium using proline as the only nitrogen source (C source is 2% glucose), and then 30 ° C. And the degree of growth was compared.
As a result, it was confirmed that the wild strain (XUW-TRP) grew using proline as the only N source, but could not grow on the strain (XUWDput1) in which PUT1 encoding proline oxidase was destroyed.

2) Genomic PCR;
Chromosomal DNA was prepared from the Trp ⁺ colony obtained by the method described above, and the destruction of PUT1 was confirmed by PCR using each primer. When the primer inside TRP1 was used on one side, amplification was not observed in XUW-TRP (wild type strain), but a specific band was amplified in XUDput1 (PUT1 disruption strain) (lane (3); 2,750 bp) , Lane (5); 1,930 bp), PUT1 destruction was confirmed.

3) proline oxidase activity;
Next, a cell extract was prepared from each strain, and proline oxidase activity was measured. Table 5 shows the measurement results. As a result of the measurement, the proline oxidase activity was detected in the wild strain (XUW-TRP) in any medium. In particular, the activity value increased about 3-fold in the proline-added medium, and it was confirmed that the expression of PUT1 was induced by proline. On the other hand, almost no activity was detected in the PUT1-disrupted strain (XUDput1).

4) intracellular proline content;
Finally, after each strain was cultured in a minimal medium for 2 days, the intracellular proline content was measured. The measurement results are shown in Table 6. From this measurement result, almost no proline was detected in both strains in the normal SD medium. However, in the medium supplemented with proline, the PUT1-disrupted strain (XUDput1) cannot degrade proline, so proline accumulates in the cells. (About 0.7% dry weight).

  From the above results, it was confirmed that PUT1 on the chromosome was destroyed in XUDput1 strain.

b) Strengthening step of synthetic system: replacement with mutant PRO1 gene (pro1 ^D154N ) Plasmid pRS-D154NPRO1 was cleaved with restriction enzymes Hind III and Sac I, and a fragment (1.8 kb) containing pro1 ^D154N gene was recovered by electrophoresis. The plasmid pR406 (purchased from Stratagene) was ligated with a URA3-containing fragment (4.3 kb) obtained by digestion with restriction enzymes Hind III and Sac I, and then introduced into E. coli strain JM109. After extracting a plasmid incorporating mutant PRO1 from ampicillin resistant transformants and preparing a linear fragment cleaved with Xba I existing in one place in PRO1, transform XUDput1 obtained in a) above. And spread on SC-Ura plates and cultured at 30 ° C. for 2-3 days.

At this time, Ura + and AZC-resistant colonies are found in the mutant PRO1 on the plasmid that undergoes homologous recombination with the wild-type PRO1 on the chromosome of the XUDput1 strain, and the full-length plasmid containing URA3 (linear fragment) has the wild-type PRO1. A transformant inserted at the locus is obtained.
Next, in order to remove the plasmid containing wild-type PRO1 and URA3, Ura + and AZC-resistant colonies were cultured in 1 ml of YPD medium at 30 ° C for 1 day, then seeded with 5% in fresh YPD medium, and then for 1 day. After culturing, the culture broth was spread on a 5-FOA-containing plate without dilution.

c) Process for selecting and separating strains that accumulate proline in cells The 5-FOA resistant colonies thus obtained are streaked in an SD medium containing AZC (100 μg / ml), and the AZC resistant colonies are mutated in the PRO1 gene (pro1 ^D154N ). Was selected as a strain remaining on the chromosome and named as XUDput1-MT, and this transformed yeast (XUDput1-MT) was deposited as FERM P-20171 at the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary.

  On the other hand, a colony showing AZC sensitivity was selected as a strain in which wild-type PRO1 remained on the chromosome and named XUDput1-WT. Furthermore, PRO1 fragments of both strains were prepared by PCR and confirmed to be the target nucleotide sequences by direct sequencing.

In this example, substitution to mutant PRO1 (pro1 ^D154N ) and intracellular proline content were examined.
The susceptibility to AZC of the strain (XUDput1-MT) and the wild strain (XUDput1-WT) in which PRO1 on the chromosome of the PUT1-disrupted strain was replaced with mutant PRO1 (pro1 ^D154N ) was compared. As a result, XUDput1-WT could not grow on SD medium containing AZC, but showed sensitivity, but it could grow on XUDput1-MT. Further, when the intracellular proline content of both strains was measured, a large amount of proline was accumulated in XUDput1-MT in relation to the growth in AZC medium (Table 7).

In this example, introduction of multiple copies of the PRO2 gene was examined.
In the FH515 strain, the stability of γ-GK and γ-GPR was improved by γ-GK amino acid substitution (D154N), so the apparent specific activity of both activities increased, and it was thought that proline was accumulated in the cells. Thus, in the laboratory strain (INVSc1), the genes of both enzymes were expressed in multiple copies and the enzyme activity was increased by the gene dosage effect, and the intracellular proline content was successfully increased.

  For the purpose of examining whether the same effect is obtained in sake yeast, the plasmid pTV-PRO2 was digested with the restriction enzyme BamH I, and a 3.4 kb fragment containing the PRO2 gene was recovered from the agarose gel, and then the plasmid pUV2 was digested with BamHI. It was ligated to the site and introduced into E. coli DH5α strain. After preparing a plasmid (pUV-PRO2) in which the PRO2 gene was correctly integrated from a transformant colony showing Amp resistance, it was introduced into the XUDput1-MT strain, and the intracellular proline content was measured. The measurement results are shown in Table 8. From this measurement result, the influence on proline content was examined by changing the culture medium and culture method, but introduction of multiple copies of PRO2 was not effective.

In this example, the laboratory yeast and the proline-accumulating yeast of the present invention were examined for growth in the presence of ethanol, ethanol stress sensitivity was examined, and the measurement results are shown in Table 9.
In laboratory yeast (MB329-17C, FH515), it was found that FH515 accumulates proline by amino acid substitution (D154N) of γ-GK and has a high survival rate in the presence of ethanol.

  In the presence of ethanol (9%, 18%), the proline-accumulating strain (XUDput1-MT (pUV-PRO2)) tended to have a higher survival rate than the pair (XUW-TRP (pUV2)). Overall, each strain had a higher survival rate in the presence of 9% ethanol than the ethanol-free medium.

In this example, a small amount of sake was prepared using a proline-accumulating strain (XUDput1-MT (pUV-PRO2)) and a control strain (XUW-TRP (pUV2)) (performed at Fukui Food Processing Research Institute). . Table 10 shows the rice used for preparation.
The strains used for the small preparation test were strains in which multiple copies of PRO2 were introduced into the strain in which wild type PRO1 was replaced with mutant PRO1 (pro1 ^D154N ) (XUDput1-MT (pUV-PRO2)), and XUW-14 strain as a control A strain (XUW-TRP (pUV2)) in which trp1 was complemented with pRS414 (TRP1) and ura3 was complemented with pUV2 (URA3) was used.

  Since there is a difference in the amount of proline accumulated in the cells depending on the culture method, both strains are pre-cultured in YPD medium (30 ml, 30 ° C, 2 days) by two methods of shaking or standing, and then n = 5 (One of them for sampling) The total rice was 200 g, the feed rate was 20%, the pumping rate was 130%, and the fermentation temperature was 15 ° C.

  After the small preparation test, each ingredient in sake was analyzed and brewing characteristics were compared. The measurement results are shown in Table 11.

  As is clear from Table 11, regarding the degree of sake (SM value) representing the specific gravity of sake relative to water, the proline-accumulating strain (XUDput1-MT (pUV-PRO2)) is better than the control strain (XUW-TRP (pUV2)). Also showed a remarkably low value. The specific gravity of sake increases as the amount of extract, mainly sugar, increases (heavier), and the degree of sake tends to minus (-). On the other hand, the smaller the extract, the smaller (lighter), and the degree of sake tends to be positive (+). In general, as the fermentation progresses, the sugar content decreases and the amount of ethanol increases, so the specific gravity decreases and the sake level increases. However, there were no changes in basic brewing characteristics such as carbon dioxide loss, ethanol production, and glucose consumption, which are indicators of fermentation, between the proline-accumulating strain and the control strain.

  Next, the amino acid content in sake was measured. As a result, the proline content in the bacterial cells in the proline-accumulating strain increased as expected (shaking: about 2-fold increase (0.13% → 0.25% dry weight), standing: about 2-fold increase (0.19% → 0.31% Dry weight), the proline content in sake was also significantly increased (shaking: 4.7 times increase (196 → 920 mg / L), standing: 5.2 times increase (171 → 883 mg / L). In the accumulating strains, amino acids such as aspartic acid, threonine, and leucine also increased, and as a result, the total amino acid content also increased by about 30% (about 1 g / L increase). Decreased.

  In addition, when comparing the organic acid content, proline accumulating strains have increased organic acids that are preferred for sake such as citric acid, malic acid (fresh acidity), and succinic acid (umami). The amount of acetic acid considered decreased. The overall organic acid content tended to increase in the proline accumulating strain (approximately 100-200 mg / L increase).

  In addition, aroma components (higher alcohols, esters, aldehydes, etc.) were also measured, but no significant difference was found between the two strains. Moreover, simple sensory evaluation was performed about the sake produced from both strains. As a result, there was a difference in flavor and taste between the two, and the proline-accumulating strain was evaluated as having a relatively soft (light) taste and flavor and easy to drink. From the above results, the proline-accumulating strains had a markedly increased proline content in sake and in the cells, and the total amino acid and organic acid contents also increased.

  Yeast is subjected to stresses such as high-concentration ethanol and low temperature in the sake production environment, and when subjected to these stresses for a long time, many intracellular proteins are denatured and the useful functions (ethanol production ability) of yeast are limited. . However, the yeast of the present invention accumulates a large amount of proline in the cell and releases a part of it to the outside of the cell. By the way, as described above, proline belongs to a sweet amino acid and has an action of protecting the yeast from ethanol, and the yeast of the present invention has the brewing characteristics of conventional yeast (glucose consumption, ethanol production, carbon dioxide gas). (Such as weight loss) is maintained as it is, and sweet sake with a high proline content can be produced efficiently. Further, since the proline-accumulating transformed yeast of the present invention produces many prolines, it is useful not only for sake, but also for the production of sweet fermented foods and brewed foods.

It is explanatory drawing regarding the metabolic pathway of proline in yeast (Saccharomyces genus cerevisiae). It is explanatory drawing regarding preparation of the PUT1 destruction strain of sake yeast. It is explanatory drawing regarding substitution to a mutant type PRO1 gene (pro1 ^D154N ).

Claims (3)

A method for producing a proline-accumulating transformed yeast having the ability to secrete a portion of proline accumulated in cells to the outside during brewing, using sake yeast comprising the following steps.
a) a step of suppressing a proline degrading system consisting of destroying a gene of a proline oxidase that is a proline degrading enzyme in a proline metabolic pathway in yeast,
b) In the yeast proline metabolic pathway, the step of strengthening the proline synthesis system consisting of substituting Asn of Asp154 of the gene encoding γ-glutamate phosphorylase, a proline synthase, and c) proline in the cell A process of selecting and separating the accumulated strains.   A proline-accumulating transformed yeast XUDput1-MT (FERM P-20171) obtained by the method according to claim 1. A method for producing sake, wherein brewing is performed using the proline-accumulating transformed yeast according to claim 2.

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